Avraham Mayevsky

June 3rd & 4th 2011

mayevsa@mail.biu.ac.il, mayevskya@gmail.com

Mitochondrial NADH and Tissue viability In Vivo:From Animal experiments to clinical Applications.

The Mina and Everard Goodman Faculty of Life-Sciences andThe Leslie and Susan Gonda Multidisciplinary Brain Research Center

Bar-Ilan University, Ramat-Gan, 52900, Israel

Britton Chance: His Life, Times, and Legacy

University of Pennsylvania, Philadelphia, USA

The Book of GenesisChapter 1,3

“And God said, Let there be light: and there was light."

Chapter 1,4“And God saw the light, that it was good: and God

divided the light from the darkness.”

(Bible-Old Testament)

The created light is helping us to shed new light into the darkness of Mitochondrial Functions

The use of light in studying mitochondrial function in vivo was introduced by my Post-Doc Mentor and teacher

Prof. Britton Chance more than 50 years ago

The letter that changed the scientific activities of my life

Short History –Monitoring of Mitochondrial function and Tissue Energy Metabolism .

“There is no instance in which it can be proven that an organ increases its activity under physiological conditions, without also increasing in its call for oxygen, and- in no organ excited by any form of stimulation can it be shown that positive work is done without the blood supply having to respond to a call for oxygen”.

Barcroft J. The Respiratory Function of the Blood.Cambridge Univ. Press, Cambridge, 1914

Mitochondrial NADH (Fluorometry)

ATP

Typical Examples:

Kidney Function

Gastrointestinal Activity

Muscle Contraction

Brain Ionic Homeostasis

Glandular Secretion

Tissue Blood Flow (LDF)

HbO2

Hemoglobin Oxygenation (Oximetry)

Venule

Arteriole

O2

O2

O2

O2

2

O2

O2O2

O2

O2O2

O2

O2

O2

O2

O2

O2

O2

Year Discovery Author(s)

1905 Involvement of adenine containing nucleotides in yeast fermentation

Harden & Young (1906)

1935 Description of the complete structure of "Hydrogen transferring Coenzyme” in erythrocytes

Warburg et al (1935)

1936 Definition of the two cofactors DPN and TPN

Warburg O (1949)

1951 A shift in the absorption spectrum of DPNH with Alcohol dehydrogenase

Theorell & Bonnichsen (1951)

1951 Development of a rapid sensitive Spectrophotometer

Chance & Legallias (1951)

Mayevsky and Rogatsky 2007

Milestones in biophotonics of Mitochondrial NADH (1)

1952 Monitoring of pyridine nucleotide enzymes Chance1957 The first detailed study of NADH using

Fluorescence spectrophotometer Duysens & Amesz

1958 Measurement of NADH fluorescence in isolated mitochondria

Chance & Baltscheffsky

1959 Measurement of muscle NADH fluorescence in vitro

Chance & Jobsis

1962 In vivo monitoring of NADH fluorescence from the brain and kidney

Chance et al

1965 Comparison between NADH fluorescence in vivo and enzymatic analysis of tissue NADH

Chance et al.

1968 Monitoring tissue reflectance in addition to NADH fluorescence

Jöbsis & Stansby

1971 The first attempt to monitor the human brain during a neurosurgical procedure

Jöbsis et al.

Milestones in Biophotonics of Mitochondrial NADH (2)

1973 The first fiber optic based fluorometer-reflectometer used in the brain of an awake animal

Chance et al.; Mayevsky & Chance

1982 Simultaneous monitoring of NADH in vivo in four different organs in the body

Mayevsky & Chance

1985 Monitoring of brain NADH together with 31P NMR Spectroscopy

Mayevsky et al.

1991 Simultaneous real time monitoring of NADH , CBF, ECoG, and extracellular ions in experimental animals and in the neurosurgical operating room

Mayevsky et al.

1996 The multiparametric response (including NADH) to cortical spreading depression is for the first time measured in a comatose patient

Mayevsky et al.

2000 Development of the FDA-approved “Tissue Spectroscope” medical device for real-time monitoring of NADH and tissue blood flow

Mayevsky et al.

2006 Monitoring of tissue vitality (NADH, TBF and HbO2) by a new “CritiView“ device

Mayevsky et al.

Milestones in biophotonics of Mitochondrial NADH (3)

The first Fiber optic based Time-Sharing Fluorometer/Reflectometer

Mayevsky and Chance 1973

Operating Room

ICU

Clinical monitoring of NADH using the CritiView -2006

A dream came through

Mitochondrial Function and NADH fluorescence measurements

The definition of mitochondrial metabolic state in 1955, by Chance and Williams, opened up a new era in spectroscopic measurements of respiratory chain enzyme’s redox state In Vitro as well as In Vivo.

NADH Oxidation-Reduction State is the

best parameter for evaluating Mitochondrial

Function In Vivo

Chance et al in 1973 concluded that “For a system in a steady state, NADH is at the extreme low potential end of the chain, and this may be the oxygen indicator of choice in isolated

mitochondria and tissues as well.”

Chance, B., Oshino, N., Sugano, T., Mayevsky, A., 1973. Basic principles of tissue oxygen determination from mitochondrial signals. In: Internat. Symposium on Oxygen Transport to Tissue, Adv. Exp. Med. Biol. Vol.37A, pp.277-292. Plenum

Pub Corp, New York ,

Why NADH ???

Scientific background underlying NADH fluorescence measurements

G.T.G.T.

GlucoseGlucose

Glycolysis

Pyruvate

OxygenOxygen

OxygenOxygen

2 ATP

H+

Lactate

Lactate

M.C.T.

TCATCA

COCO22H+

OO22

HH22OO

NADNAD++ NADHNADH

ADP+Pi

ATP36

ATP

GlucoseGlucose O2

OO22

G.T.G.T.

GlucoseGlucose

Glycolysis

Pyruvate

OxygenOxygen

OxygenOxygen

2 ATP

H+

Lactate

Lactate

M.C.T.

TCATCA

COCO22H+

OO22

HH22OO

NADNAD++ NADHNADH

ADP+Pi

ATP36

ATP

GlucoseGlucose O2

OO22

Principles of Tissue Energy Metabolism In Normal cells

100

20-30

1

95

0

50

100

150

Alveoli Arterial Blood

AIR Tissue Intramitochondrial

Ox y

gen

Par ti

al P

r ess

ure

(mm

Hg)

160N2

O2End Tidal

CO2 Heart Rate &ECG

Cardiac Output

Systemic Blood Pressure

Systemic Saturation(Pulse Oximetry)

CritiViewMicrocirculation blood flow and

oxygenationNADH redox state

The Mitochondrion

The NADH molecule is a control marker in the energy generation chain in the mitochondria

An increase in the NADH levels indicates that metabolic imbalance unfolds

Am. J. Physiol. Cell Physiol. 292: C615-C640 )2007( .

A. NADH - The Mitochondrion “Flag”

B. Absorption Spectra of NAD+ and NADH

C. NADH Fluorescence spectra

nm

y = 0.0191x + 0.3529R2 = 0.9945

y = 0.0195x + 0.4522R2 = 0.9918

0

1

2

3

4

5

6

7

0 100 200 300 400NADH(mM)

Crit

iVie

w (V

olts

)

set#1set#2

NADH Calibration in Solution

Methods and Technology used in the past and current state of art

From Single parameter to multiparametric monitoring approach

Science, 137:499-508, 1962

F.F. Jobsis Group Fluorometer 1970th

B.Chance Fluorometer 1960th

Am. J. Physiol 243: H619-627 (1982)

Mayevsky A. Brain Res. Rev. 7: 49‑68, )1984( .

Various Types of Fluorometers Developed During 1970-1980

Effects of Anoxia (100% Nitrogen) - Brain

NADH Oxidation

Effects of Cortical Spreading Depression on Brain NADH

A. Mayevsky, D. Jamieson and B. Chance, Brain Res. 76, 481-491 )1974(.

Effects of Hyperbaric Oxygenation on brain NADH and EEG

Mitochondrial Redox state In Vitro and Brain NADH Responses In Vivo

A

B

B. Chance, A. Mayevsky, C. Goodwin and L. Mela, Microvasc. Res. 8, 276-282 )1974(.

M Osbakken et alJ. Appl. Cardiol. 4: 305‑313 )1989(.

Diagram of the light guide, used in conjunction with a fluorometer built in our laboratory, and the surface coil on heart. HV = high voltage, PM = photomultiplier tubes.

Monitoring the Beating Heart In Vivo

J. Appl. Cardiol. 4: 305‑313 )1989(.

Typical NADH responses of dog myocardium during (A) hypoxia and (8) pressure loading. AOP = aortic pressure, CF = corrected fluorescence. F = fluorescence, PAP = pulmo- nary artery pressure, R = reflectance, VP = ventricular pressure. Note that the NADH response to norepinephrine was related to maximal NADH response to hypoxia (in this case, anoxia produced by using 100% inspired N2.

Low Temperature Scanning of NADH and Fp in Frozen Tissues

Chance et al 1978

Brain Res. 367: 63-72 )1986(.

Effects of right carotid occlusion on the redox states measured in two brain depths.

Scanning of NADH and Fp in the Partial Ischemic Brain

Fig. 3. Four-channel DC fluorometer/reflectometer connected to the gerbil brain using a flexible fiber optic bundle (for details see text). Brain Res. Rev. 7: 49‑68, )1984(.

Multichannel Monitoring of NADH Redox State In Vivo

(A) Effects of graded hypoxia and anoxia on the NADH redox state in an artificially,--- ventilated rat. Four organs were monitored simultaneously, and for each organ we recorded the reflectance (R) and the corrected fluorescence (CF). Subscripts: B, brain; L, liver; K, kidney; and T, testis. (B) Effects of asphyxia.

Brain

Liver

Kidney

Testis

Science 217, 6 August ,1982.

Multiorgan Monitoring of NADH Redox State in the Rat

A. Mayevsky, S. Lebourdais and B. Chance, J. Neurosci. Res. 5, 173-182 )1980(.

A B

A. Mayevsky, K. H. Frank, S. Nioka, M. Kessler and B. Chance, in Oxygen Transport to Tissue XII, J. Piiper, T. K. Goldstick, M. Meyer, Eds., pp. 303-313, Plenum Press, )1990(.

A. Mayevsky, D. Jamieson and B. Chance, Brain Res. 76, 481-491 )1974(.

A. Mayevsky, S. Nioka, D. J. Wang and B. Chance, in Oxygen Transport to Tissue XVIII, E. M. Nemoto and J. C. LaManna, Eds., pp. 41-53, Plenum Press, )1997(.

A. Mayevsky, S. Nioka, D. J. Wang and B. Chance, in Oxygen Transport to Tissue XVIII, E. M. Nemoto and J. C. LaManna, Eds., pp. 41-53, Plenum Press, )1997(.

A. Mayevsky, E. S. Flamm, W. Pennie and B. Chance, "A fiber optic based multiprobe system for intraoperative monitoring of brain functions," SPIE Proc. 1431, 303-313 )1991(

The CritiView Device, Probes and Clinical Applications

Open Chest Heart Surgery

38min

CABG

In this patient the hemodynamic and mitochondrial responses started very early in the operation procedure.

Chest open

Pump on

Chest closure

Pump off

GS942 22 JAN 2007- 15H40M

22min

In this patient clear responses to the procedure were recorded. At 16:49, the pump ON condition led to a large decrease in TBF as well as a large increase in NADH. The signals returned toward the initial values although base line was not reached )monitoring period ends at 18:14(

kidneyTestis

Small Intestine

Liver

Heart

Urethra

Spinal cord

Animal

ClinicalClinicalPigs

IschemiaNE

NE

IschemiaNE

HypercapniaPapaverineIschemia

N2

NE Hemorrhage AAAICU

Bypass

PacingHypopneaIschemia

Drugs (Ach, NE, vasoactive)

CompressionIschemia

Brain

Oxygen deficiencyIschemia

NO

Drugs

TBI

HyperbariaHBO

Clinical

Activation

CO

Hemorrhage

HypothermiaAging

Sepsis

EpilepsySD

MannitolICP elevation

Retraction

AnoxiaHypoxia

Hypercapnia

NimodipineEthanol

AnestheticsUncoupler

During operationICU

MitochondrionVolume 1, Issue 1 ,June 2001, Pages 3-31

Review articleA century of mitochondrial research: achievements and perspectives

Immo E. Scheffler

Out of 247 References Only one Reference By Chance was cited

Science, 137:499-508, 1962

Mayevsky, A. and Chance, B. Mitochondrion 7: 330-339 )2007(.

Effects of Adrenaline on various organs

Thank you for the attention